Hematopoietic stem cells (HSCs) can self renew and generate all lineages of the hematopoietic system. Despite significant progress in our understanding of mechanisms involved in self-renewal, differentiation and quiescence of HSCs, a coherent picture of how these mechanisms act in concert to regulate homeostatic responses of the hematopoietic system in vivo has not emerged yet. Several transcriptional regulators involved as partners of leukemogenic fusion proteins play a critical role in HSC biology. We found, using germline Prdm16-/- mice, that Prdm16, a 140kDa zinc finger protein that was originally discovered as a fusion partner in some translocations in acute myeloblastic leukemia (AML), is essential for the establishment and maintenance of HSCs during development and after transplantation. In this proposal we want to further analyze the function of Prdm16 in the biology of HSCs, and determine its mechanism of action. Our preliminary data strongly suggest a role for Prdm16 in maintaining mitochondrial function and integrity specifically in HSCs. Prdm16 occurs in two splice forms. Short (s) Prdm16 lacks the N-terminal PR domain. This splice variant is typically overexpressed in leukemias through translocation, promoter hypomethylation, or retroviral insertion into the PR domain. Our preliminary data indicate that primarily sPrdm16 is responsible for the hematopoietic phenotype of Prdm16-deficient mice. The specific aims are the following: Aim 1: To determine the critical developmental window of Prdm16 requirement for HSC function; Aim 2: To examine metabolism, mitochondrial function, and dynamics in Prdm16-/- HSCs and MEFs; Aim 3: To examine the roles of sPrdm16 vs. flPrdm16 in HSC maintenance.